Investigation Of The Mechamisms Of The Intronic 27-nt MiRNA In ENOS Expression

Objective To investigate the mechamisms of the polymorphic intronic 27-nucleotide(nt) microRNAs(miRNAs) in endothelial nitric oxide synthase(eNOS) expression.Methods Using the synthetic double-strand 27 base pair (bp) DNA, which is based on the sequence of the 27 nucleotide (nt) intronic miRNA, cloned the miRNA highly expression plasmid by inserting the 27bp DNA into the vector of pSilencerTM 3.1- H1 neo. After the plasmid confirming by sequencing, the 27bp miRNA plasmid were then transferred into HUVEC with lipofectamine 2000, consequently cell line with the 27-nt miRNA highly expression was established by screening with G418. Northern bloting was used to verify the miRNA expression in the stable cell lines. The level of eNOS,nuclear transcription factor stimulating protein-1(SP-1),activatorprotein-1( AP-1 ) ,signal transducers and activators of transcription3(STAT3),vascular endothelial growth factor (VEGF) mRNA and protein were measured by RT-PCR and Western blotting, respectively.Results The results of the sequencing showed that there was no reading frame shifts and mutations in recombinant vector of 27bp, which suggested that the plasmid was successfully constructed. The RT-PCR and Western blotting results showed that both at mRNA and protein level,overexpression of 27-nt miRNA and its duplexes and mutants can inhibited eNOS expression at different levels . However, our four miRNAs can,t influence the expression of VEGF.Transcription factor SP-1 expression was suppressed only in the cells overexpressing 54-nt and its mutant miRNA. AP-1 expression was reduced in the cells overexpressing both 27-nt and 54-nt miRNA(including wide-type and mutant) at mRNA and protein level. STAT3 expression was reduced in the cells overexpressing 27-nt mutant and 54-nt mutant miRNA at mRNA level and didn't express in the cells overexpressing 54-nt and its mutant miRNA at protein level.Conclusion1. Intronic 27-nt miRNA significantly inhibit the eNOS mRNA and protein expression,which is correlative with miRNA polymophisms.2. Transcription factors SP-1,AP-1 and STAT3 are involved in the regulation of eNOS expression by the 27-nt miRNA.3. Polymorphic intronic 27- nt miRNAs can,t influence the expression of VEGF.